Bacterial biofilms are highly heterogeneous and found in the natural, industrial, and medical environments and include microorganisms embedded in a glycocalyx that is predominantly composed of microbially produced exopolysaccharide (Flemming et al., in “Biofilms: recent advances in their study and control”, 2000, pp. 19-34, Harwood Academic Publishers, Amsterdam, The Netherlands; Costerton et al., Science, 1999, 284:1318-1322; Costerton et al., J. Bacteriol., 1994, 176:2137-2142; Keevil et al., Microbiol. Eur., 1995, 3:10-14). The glycocalyx can provide protection against environmental change, such as antimicrobial agents, and may act as a reservoir for nutrients and ions (Allison, Microbiol. Eur., 1993, Nov./Dec. 16-19; Mah et al., Trends Microbiol., 2001, 9:34-39; Stewart and Costerton, Lancet, 2001, 358:135-138).
The presence of persistent bacterial biofilms is known to contribute to the molecular pathologies of many diseases such as periodontal disease, cystic fibrosis, and chronic otitis media, as well as infections associated with contact lenses, urinary catheters, central venous catheters, endotrachael tubes, and surgical devices (R. M. Donlan and J. W. Costerton, Clin. Microbiol. Rev. 15, 167-193 (2002); J. W. Costerton, P. S. Stewart, E. P. Greenberg, Science 284, 1318-1322 (1999)). There has been increased recognition that bacterial colonization, particularly the presence of microbial biofilm, is one of the main factors causing delayed wound healing (S. G. Jones, R. Edwards, D. W. Thomas, Int. J. Low Extrem. Wounds 3, 201-208 (2004); Edwards, R., Harding K G (2004) “Bacteria and Wound Healing” Curr Opin Infect Dis 17:91-96; and James, G A, Swogger, E. Wolcott R, Pulcini E., Secor P., Sestrich, J. Costerton, J. W., Stewart, P. S. (2008) “Biofilms in Chronic Wounds” Wound Repair Regen 16:37-44). Recent investigations indicate that most chronic skin wounds (˜60%) contain bacterial biofilms and a small percentage of acute wounds (6%) clearly have bacterial biofilms (James et al., 2008, ibid.).
Bacteria embedded in biofilms are physiologically different from planktonic (free-floating) ones. Regardless of location or diversity, all microbial biofilms have a common developmental process including attachment, colonization, maturation, and dispersion (R. M. Donlan and J. W. Costerton, Clin. Microbiol. Rev. 15, 167-193 (2002); J. W. Costerton, P. S. Stewart, E. P. Greenberg, Science 284, 1318-1322 (1999); M. E. Davey and G. A. O'Toole, Microbiol. Mol. Biol. Rev. 64, 847-867 (2000); A. L. Spoering and M. S. Gilmore, Curr. Opin. Microbiol. 9, 133-137 (2006); and P. Hunter, EMBO Rep. 9, 314-317 (2008)). Planktonic bacteria reversibly adhere to surfaces, such as open wounds or medical devices, and/or coaggregate (specific bacterial cell-to cell attachment) at surface interfaces (air-water). In response to environmental signals, they become sessile (irreversibly attached) and secrete a protective matrix consisting of self-synthesized extracellular polymeric substance (EPS), then differentiate and form microcolonies, and finally build up to form complex three dimensional biofilms (J. W. Costerton, P. S. Stewart, E. P. Greenberg, Science 284, 1318-1322 (1999); D. G. Davies et al., Science 280, 295-298 (1998); P. Gilbert, J. Das, I. Foley, Adv. Dent. Res. 11, 160-167 (1997); and R. M. Donlan and J. W. Costerton, Clin. Microbiol. Rev. 15, 167-193 (2002)).
The structural complexity of biofilms is thought to be analogous to tissues of higher organisms. Biofilm structures consist of interstitial channels and distinctly located subpopulations of cells with different patterns of gene expression (P. Watnick and R. Kolter, J. Bacteriol. 182, 2675-2679 (2000)). These structural features are believed to allow the dense cell populations in biofilms to overcome the potential limitation of nutrients and oxygen, enable exchange of metabolic products and signal molecules, and facilitate removal of toxic metabolic products and waste (K. D. Xu, P. S. Stewart, F. Xia, C. T. Huang, G. A. McFeters, Appl. Environ. Microbiol. 64, 4035-4039 (1998); M. R. Parsek and E. P. Greenberg, Trends Microbiol. 13, 27-33 (2005); D. de Beer, P. Stoodley, Z. Lewandowski, Biotechnology and Bioengineering 44, 636-641 (2004); D. de Beer, P. Stoodley, F. Roe, Z. Lewandowski, Biotechnology and Bioengineering 43, 1131-1138 (2004); J. W. Costerton, Z. Lewandowski, D. E. Caldwell, D. R. Korber, H. M. Lappin-Scott, Annu. Rev. Microbiol. 49, 711-745 (1995); and P. Stoodley, D. Debeer, Z. Lewandowski, Appl. Environ. Microbiol. 60, 2711-2716 (1994)).
Compared to planktonic bacteria, coaggregated surface attached (sessile) microcolonies of bacteria in biofilms such as those found in chronic skin wounds have enhanced resistance to killing by endogenous antibodies and phagocytic cells, as well as by exogenous antibiotics, antiseptics, and disinfectants (J. W. Costerton, P. S. Stewart, E. P. Greenberg, Science 284, 1318-1322 (1999); R. M. Donlan and J. W. Costerton, Clin. Microbiol. Rev. 15, 167-193 (2002); R. Edwards and K. G. Harding, Curr. Opin. Infect. Dis. 17, 91-96 (2004); and J. G. Leid et al., J. Immunol. 175, 7512-7518 (2005)). This has lead to the more recent appreciation of the need for reassessing the efficacy of conventional antimicrobial treatments and the need to develop new treatment strategies specific for managing microbial biofilm in wounds, particularly in chronic wounds.
Chronic wounds recalcitrant to healing are an increasingly grave worldwide problem. They include diabetic foot ulcers (DFU), pressure ulcers (PU), and venous leg ulcers (VLU), which lead patients to chronic pain, impaired mobility, frequent amputations, and reduced life quality. More than 1% of the population in developed countries has been estimated to experience a chronic wound during their lifetime, an occurrence that is increasing with the number of lifestyle diseases such as obesity, diabetes, and cardiovascular diseases (F. Gottrup, M. S. Agren, T. Karlsmark, Wound Repair Regen. 8, 83-96 (2000)). In 2000, diabetes was estimated to affect 171 million people worldwide and is predicted to more than double by 2030 (G. A. Matricali, G. Dereymaeker, E. Muls, M. Flour, C. Mathieu, Diabetes Metab Res. Rev. 23, 339-347 (2007)). Approximately 15% of diabetic patients will develop lower extremity ulcers and 14-24% of DFU will eventually undergo amputation (G. E. Reiber, Diabet. Med. 13 Suppl 1, S6-11 (1996); and G. E. Reiber et al., Diabetes Care 22, 157-162 (1999)). 1% of the world's population suffer with VLU (J. T. Trent, A. Falabella, W. H. Eaglstein, R. S. Kirsner, Ostomy Wound Manage. 51, 38-54 (2005)).
The expenditure on chronic wounds is enormous and a financial toll worldwide. In 2004, the total cost of DFU rose to $10 billion, including direct expenses (about 4% of the total personal health spending) and another $5 billion in indirect expenses (disability, nursing homes, etc.). The majority of the direct cost of DFU (71-88%) is attributed to in-hospital stay (length of stay being the most important factor) while the single contribution of other factors (drugs, investigations, surgery, orthopedic appliances, visits to foot care specialists, home care) is comparatively low (>10%) (G. A. Matricali, G. Dereymaeker, E. Muls, M. Flour, C. Mathieu, Diabetes Metab Res. Rev. 23, 339-347 (2007)). Medicare reimbursement remains insufficient, with hospital costs exceeding reimbursement by almost $7500 per patient (Matricali et al., 2007, ibid.).
The center of disease control (CDC) estimates that among all nosocomial infections, biofilm-based infections contribute more than 65% (C. Potera, Science 283, 1837-+ (1999)) which has lead to an increase in patients' hospitalization by 2 to 3 days and additional costs of over $1 billion per year (L. K. Archibald and R. P. Gaynes, Infect. Dis. Clin. North Am. 11, 245-255 (1997)). As mentioned previously, the presence of microbial biofilm is one of the main factors believed to cause delayed wound healing (R. Edwards and K. G. Harding, Curr. Opin. Infect. Dis. 17, 91-96 (2004); S. G. Jones, R. Edwards, D. W. Thomas, Int. J. Low Extrem. Wounds 3, 201-208 (2004); and G. A. James et al., Wound. Repair Regen. 16, 37-44 (2008)). In the U.S. alone, chronic wounds affect over 4 million people with treatment costs of $9 billion per year (K. Izadi and P. Ganchi, Clin. Plast. Surg. 32, 209-222 (2005)). As a consequence, chronic wound healing is of significant importance to human health as well as economic development.
Unfortunately, there are currently no topographical wound assessment devices for the detection of wound biofilm or microorganisms. In addition, there is a need for rapid point-of-care devices for detecting wound bacteria and/or bacterial biofilm. The conventional method of diagnosing the presence of microorganisms (bacterial and fungal) in wounds is technologically complex and time consuming, involving sampling, culturing, and typing in clinical microbiology labs. This procedure can cause significant delays in assessing the condition of the wound and administering appropriate treatment.
In addition to the delay in administering appropriate treatment to patients due to the time required analyzing samples, it has been well documented that biofilm in chronic wounds contain a number of uncultivable and difficult to culture species (P. G. Bowler and B. J. Davies, Int. J. Dermatol. 38, 573-578 (1999); C. E. Davies et al., J. Clin. Microbiol. 42, 3549-3557 (2004); and S. E. Dowd et al., BMC. Microbiol. 8, 43 (2008)), making the characterization of the wound microflora and identification of potential pathogens or primary contributors to pathology difficult. This basic deficiency in diagnosis often results in ineffective treatment strategies.
Thus, there is a need for a rapid, simple, inexpensive, point-of-care assay that would detect and localize bacterial biofilms and/or microorganism in chronic wounds in order to develop more effective treatment strategies in wound management.